Presentation Information
[P02-278]Multiscale Modelling Guides Rhodopsin Spectral Design and Reveals Pathway-Selective Growth Advantages in Formatotrophic Cupriavidus necator
○Haris Saeed1, Wei Huang1, Aidong Yang1 (1. University Of Oxford (UK))
Keywords:
Metabolic Modelling,C1 Conversion,Sustainable Bioprduction
[Purpose]
Engineering microbes for efficient C1 conversion requires tight coupling of energy supply and carbon metabolism. Engineered photosynthetic chemolithoautotrophs offer a promising route, yet no quantitative model currently connects the spectral design of a light harnessing to the specific metabolic constraints that it alleviates. Here, we develop and partially experimentally validate a multiscale model for predicting how expression of teh light driven proton pump-rhodopsin, and metabolic engineering reshapes growth in formatotrophic Cupriavidus necator.
[Method]
We model Rhodopsin using wavelength-resolved quantum yield, inferred using Zhu–Nakamura trajectory surface-hopping and Markov chain Monte Carlo calibration against experimental data, with seven-state photocycle kinetics to link spectral effects to photocycle dynamics. This is then linked to enzyme-constrained flux balance analysis based on a genome-scale metabolic model of the chemolithoautotroph C. necator. Using this model, we compare the reductive glycine pathway (rGlyP) with the Calvin–Benson–Bassham cycle (CBB) across a range of light-driven proton pump rates, wavelengths, and osmotic stress conditions. Culture-scale optical effects are represented using a two-flux model incorporating wavelength-dependent scattering and background absorption.
[Results]
Rhodopsin expression, pathway choice, and spectral tuning emerge as three independently stackable engineering levers. In the absence of light, rGlyP already achieves approximately 23% higher biomass yield per gram of formate than CBB, reflecting its more favourable ATP:NADPH stoichiometry for formate assimilation. Rhodopsin expression provides ATP-only supplementation that relieves the ATP bottleneck in rGlyP, increasing rGlyP biomass yield by a further ~18% relative to the dark baseline, while CBB, which remains jointly constrained by both ATP and NADPH, gains only ~15%. Thus, rGlyP with optimised rhodopsin expression achieves approximately 45% higher biomass yield per gram of formate than CBB without rhodopsin. Spectral tuning towards 520–540 nm further amplifies the per-molecule pump rate, with the net benefit scaling from ~6% at low light intensities to ~50% under saturating conditions. The full benefit is realised in optically thin or well mixed reactor configurations.
[Consideration]
The photophysical calibration relies on wild-type measurement alone, resulting in broad credible intervals at non-native wavelengths. The model assumes metabolic and photophysical steady states, and therefore does not capture dynamic light gradients or substrate fluctuations.
[Conclusion]
These results identify rGlyP as the preferred pathway for rhodopsin-supplemented formatotrophic production, and blue-shifted variants as optimal in optically accessible reactor configurations. The multiscale model is generalisable to any organism in which light-driven ion pumps augment substrate-derived ATP. Or where an undertanding of dynamics at the molecular scale
Engineering microbes for efficient C1 conversion requires tight coupling of energy supply and carbon metabolism. Engineered photosynthetic chemolithoautotrophs offer a promising route, yet no quantitative model currently connects the spectral design of a light harnessing to the specific metabolic constraints that it alleviates. Here, we develop and partially experimentally validate a multiscale model for predicting how expression of teh light driven proton pump-rhodopsin, and metabolic engineering reshapes growth in formatotrophic Cupriavidus necator.
[Method]
We model Rhodopsin using wavelength-resolved quantum yield, inferred using Zhu–Nakamura trajectory surface-hopping and Markov chain Monte Carlo calibration against experimental data, with seven-state photocycle kinetics to link spectral effects to photocycle dynamics. This is then linked to enzyme-constrained flux balance analysis based on a genome-scale metabolic model of the chemolithoautotroph C. necator. Using this model, we compare the reductive glycine pathway (rGlyP) with the Calvin–Benson–Bassham cycle (CBB) across a range of light-driven proton pump rates, wavelengths, and osmotic stress conditions. Culture-scale optical effects are represented using a two-flux model incorporating wavelength-dependent scattering and background absorption.
[Results]
Rhodopsin expression, pathway choice, and spectral tuning emerge as three independently stackable engineering levers. In the absence of light, rGlyP already achieves approximately 23% higher biomass yield per gram of formate than CBB, reflecting its more favourable ATP:NADPH stoichiometry for formate assimilation. Rhodopsin expression provides ATP-only supplementation that relieves the ATP bottleneck in rGlyP, increasing rGlyP biomass yield by a further ~18% relative to the dark baseline, while CBB, which remains jointly constrained by both ATP and NADPH, gains only ~15%. Thus, rGlyP with optimised rhodopsin expression achieves approximately 45% higher biomass yield per gram of formate than CBB without rhodopsin. Spectral tuning towards 520–540 nm further amplifies the per-molecule pump rate, with the net benefit scaling from ~6% at low light intensities to ~50% under saturating conditions. The full benefit is realised in optically thin or well mixed reactor configurations.
[Consideration]
The photophysical calibration relies on wild-type measurement alone, resulting in broad credible intervals at non-native wavelengths. The model assumes metabolic and photophysical steady states, and therefore does not capture dynamic light gradients or substrate fluctuations.
[Conclusion]
These results identify rGlyP as the preferred pathway for rhodopsin-supplemented formatotrophic production, and blue-shifted variants as optimal in optically accessible reactor configurations. The multiscale model is generalisable to any organism in which light-driven ion pumps augment substrate-derived ATP. Or where an undertanding of dynamics at the molecular scale
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